Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C0029463 (osteosarcoma)
 16,637 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Analogs of the seco-steroid hormone, 1,25-dihydroxyvitamin D3 [1,25-(OH)-2D3] can preferentially stimulate genomic or nongenomic signaling pathways in osteoblasts. In this study, we used 1,25-(OH)2D3 analogs and voltage-sensitive Ca2+ channel (VSCC) ligands, including dihydropyridines (Bay K 8644 and nitrendipine), in an osteosarcoma cell model to examine the relationship between 1,25-(OH)2D3-stimulated Ca2+ influx and genomic and nongenomic pathways leading to osteoblast activation. Northern blotting experiments demonstrated that an analog of 1,25-(OH)2D3, 1,24-dihydroxy-22-ene-24-cyclopropyl D3, increased messenger RNA (mRNA) levels of both osteopontin (OPN) and osteocalcin (OCN) without triggering Ca2+ influx through VSCCs. Nitrendipine (an inhibitor of L-type VSCCs) did not block the mRNA increase induced by either analog 1,24-dihydroxy-22-ene-24-cyclopropyl D3 or 1,25-(OH)2D3. 1-Deoxy analogs of 1,25-(OH)2D3, 25-hydroxy-16-ene-23-yne-D3, or 25-hydroxy-23-yne-D3, which stimulate Ca2+ influx, did not produce mRNA accumulation for OPN and OCN, consistent with their poor binding to nuclear receptors. Likewise, Bay K 8644, an agonist of VSCCs that produces Ca2+ influx, did not increase mRNA levels for OPN or OCN. Experiments using a construct derived from the sequence of the genomic OPN promoter region and a luciferase reporter confirmed the analog specificity in stimulating transcription. Together these results indicate that 1,25-(OH)2D3-mediated up-regulation of genes encoding OPN and OCN is independent of Ca2+ influx and suggest that the stimulation of Ca2+ influx by 1,25-(OH)2D3 is not required for target gene activation.
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PMID:Target gene activation by 1,25-dihydroxyvitamin D3 in osteosarcoma cells is independent of calcium influx. 798 30

Choline acetyltransferase (ChAT) is specifically expressed in cholinergic neurons. To identify control mechanisms regulating the cell-specific expression of the gene encoding ChAT, transient expression of the luciferase gene driven by human ChAT gene 5'flanking sequences was compared in cholinergic and noncholinergic cell lines. Analysis of the gene indicated the presence of two regulatory elements with selective silencing activity. These elements, located between nucleotides -2043 to -3347 and nucleotides -3347 to -6550, act cooperatively to repress promoter activity > 10-fold in a human adrenergic neuroblastoma cell line, SHSY5Y, and a human osteosarcoma cell line, 143 TK-, while exhibiting less than a two-fold effect in cholinergic cell lines. Deletion of either nucleotides -2043 to -3347 or nucleotides -3348 to -6550 reduced cell-specific repression by approximately half. Such differential repression appears to be responsible for the selective expression of the ChAT component of the cholinergic phenotype.
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PMID:Cholinergic neuron-specific expression of the human choline acetyltransferase gene is controlled by silencer elements. 833 50

Bone sialoprotein (BSP), a protein which has been implicated in the initial mineralization of newly-formed bone, provides an early phenotypic marker for differentiated osteoblasts. BSP expression is induced by glucocorticoids in association with osteoblast differentiation, and a glucocorticoid response element (GRE) overlapping a putative TRE (TPA, 12-O-tetradecanoyl-phorbol 13-acetate, response element) site has been identified in the rat BSP promoter (Ogata et al., 1995). Since AP-1 and the glucocorticoid receptor have a central role in regulating cell proliferation and differentiation, we have studied AP-1 activity, stimulated by 100 ng/ml TPA in normal fetal rat calvarial cells and in transformed rat osteosarcoma cells (ROS 17/2.8). A transient induction of both c-fos and c-jun mRNAs by TPA was observed in both cell populations, together with an associated suppression of BSP mRNA in the fetal rat calvarial cells. Rat BSP promoter constructs, transiently transfected into ROS 17/2.8 cells, were used to show that TPA suppressed transcription of a luciferase construct (-938/+60; pLUC6) that included the GRE/TRE, but not transcription of shorter contructs lacking this element. Notably, suppression of pLUC6 transcription by TPA was abrogated in the presence of the synthetic glucocorticoid, dexamethasone. Gel mobility shift analyses were performed using two double-stranded synthetic oligonucleotides. These encompassed the TRE and either the distal pair of GRE half-sites (-936/ -910; GRE3) or the proximal pair of GRE half-sites (-925/-899; GRE 4) that comprise the GRE/AP-1 element. The assay showed binding of both AP-1 complexes and recombinant c-Jun homodimers. Additionally, either the c-Jun or glucocorticoid receptor could displace its counterpart from the GRE/TRE but not from consensus GRE and TRE oligonucleotides, indicating that the abrogation of AP-1-mediated gene suppression by glucocorticoids could involve competitive binding. These studies, therefore, have identified a glucocorticoid response unit through which c-Fos and c-Jun can suppress the expression of BSP in proliferating pre-osteoblastic cells and through which glucocorticoids can ameliorate the effects of AP-1 and promote osteoblast differentiation and the associated expression of BSP.
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PMID:AP-1 regulation of the rat bone sialoprotein gene transcription is mediated through a TPA response element within a glucocorticoid response unit in the gene promoter. 883 13

Transforming growth factor-beta (TGF-beta) increases steady-state mRNA levels of several extracellular matrix proteins in mineralized connective tissues. Bone sialoprotein (BSP) is a major constituent of the bone matrix, thought to initiate and regulate the formation of mineral crystals. To determine the molecular pathways of TGF-beta 1 regulation of bone proteins, we have analyzed the effects of the TGF-beta 1 on the expression of the BSP in the rat osteosarcoma cell line (ROS 17/2.8). TGF-beta 1 at 1 ng/ml, increased BSP mRNA levels in ROS 17/2.8 cells approximately 8-fold: the stimulation was first evident at 3 hr, reached maximal levels at 12 hr and slowly declined thereafter. Since the stability of the BSP mRNA was not significantly affected by TGF-beta 1, and nuclear "run-on" transcription analyses revealed only a approximately 2-fold increase in the transcription of the BSP gene, most of the increase in BSP mRNA appeared to involve a nuclear post-transcriptional mechanism. Moreover, the effects of TGF-beta 1 were indirect, since the increase in BSP mRNA was abrogated by cycloheximide (28 micrograms/ml). To identify the site of transcriptional regulation by TGF-beta 1, transient transfection analyses were performed using BSP gene promoter constructs linked to a luciferase reporter gene. Constructs that included nt -801 to -426 of the promoter sequence were found to enhance transcriptional activity approximately 1.8-fold in cells treated with TGF-beta 1. Within this sequence, approximately 500 nt upstream of the transcription start site, a putative TGF-beta activation element (TAE) was identified that contained the 5'-portion of the nuclear factor-1 (NF-1) canonical sequence (TTGGC) overlapping a consensus sequence for activator protein-2 (AP-2). The functionality of the TAE was shown by an increased binding of a nuclear protein from TGF-beta 1 stimulated cells in gel mobility shift assays and from the attenuation of TGF-beta 1-induced luciferase activity when cells were co-transfected with a double-stranded TAE oligonucleotide. Competition gel mobility shift analyses revealed that the nuclear protein that binds to the TAE has similar properties to, but is distinct from, NF-1 nuclear protein. These studies have therefore identified a TGF-beta activation element (TAE) in the rat BSP gene promoter that mediates the stimulatory effects of TGF-beta 1 on BSP gene transcription.
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PMID:Transforming growth factor-beta 1 regulation of bone sialoprotein gene transcription: identification of a TGF-beta activation element in the rat BSP gene promoter. 917

The pericellular proteoglycan biglycan is among the major secretory products of osteoblasts and articular chondrocytes but the regulatory agents and signal transduction pathways that ultimately lead to alterations in biglycan gene expression are poorly defined. We report here on the transcriptional up-regulation of biglycan in MG-63 osteosarcoma cells by agents that increase intracellular cAMP levels. Transfection of these cells with biglycan promoter luciferase reporter fusion genes and subsequent treatment with forskolin or the cAMP analog 8-Bromo-cAMP resulted in an up to 3.8-fold stimulation of biglycan promoter activity. This effect could be prevented with the compound KT5720, a specific inhibitor of the cAMP-dependent protein kinase. Up-regulation of transcription is also reflected at the level of mRNA expression, since biglycan mRNA steady state levels in MG-63 cells increased approximately 2-fold after 24 hours of forskolin treatment. These data suggest that elevated levels of intracellular cAMP increase transcription from the biglycan promoter in bone cells and implicate for the first time the cAMP/protein kinase A signal transduction pathway in the regulation of biglycan gene expression.
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PMID:Biglycan gene promoter activity in osteosarcoma cells is regulated by cyclic AMP. 919 8

p21(Waf1/Cip1) is one of the key regulatory proteins in cell cycle, terminal differentiation, and apoptosis. Its promoter was shown to be transactivated by the wild-type p53 protein as well as in a p53-independent manner. In this report, we demonstrate that E1AF, an ets-related transcription factor, activates the human p21(Waf1/Cip1) promoter by interacting with the ets-binding sites located close to the two previously identified p53-responsive elements. Northern blot analysis revealed that p21(Waf1/Cip1) and E1AF were correlatively upregulated in response to cisplatin treatment in SiHa cells. Transient expression assays demonstrated that E1AF can activate the p21(Waf1/Cip1) promoter-driven luciferase reporter gene in SiHa cells. The p21(Waf1/Cip1) promoter activity was also increased in p53-null Saos2 osteosarcoma cells, but was markedly reduced when the ets-binding sites were deleted. These results indicate that E1AF positively regulates transcription from the p21(Waf1/Cip1) promoter in response to genotoxic stresses.
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PMID:Activation of the p21(Waf1/Cip1) promoter by the ets oncogene family transcription factor E1AF. 922 30

Osteocalcin (OC) is a bone-specific extracellular matrix protein expressed by mature osteoblasts during late stages of differentiation. Previous studies have shown that forskolin, an activator of adenylate cyclase, stimulated OC production. Because PTH has been shown to activate several intracellular signal transduction pathways including cAMP, inositol phosphate and intracellular calcium mobilization, we investigated whether PTH action on cAMP accumulation leads to OC promoter activation. The rat OC promoter (1095 bp) was cloned into the promoterless luciferase gene reporter vector. The transcriptional activity of the rat OC promoter was evaluated after transfection of SaOS-2, an osteosarcoma cell line, with the OC promoter followed by treatment with PTH. Maximal OC promoter activity was observed within 4-8 h after the addition of 10(-8) M PTH, whereas very little induction was seen after 24 and 48 h of treatment. The induction of OC promoter activity by PTH was concentration dependent. PTH analogs (PTH 1-84, PTH 1-34, and PTH 1-31) that stimulate intracellular cAMP accumulation, induced OC promoter activity, whereas other PTH analogs (PTH 3-34, PTH 7-34, PTH 13-34, and PTH 53-84) that do not stimulate cAMP production had no effect on OC promoter activation. Furthermore, PTH activation of the OC promoter was significantly enhanced in the presence of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Inactivation of cAMP-dependent protein kinase A activity by either a selective protein kinase A inhibitor, H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5 isoquinolinesulfonamide), or antisense oligonucleotide directed against the regulatory subunit of cAMP-dependent protein kinase A, led to a corresponding loss of OC promoter activation by PTH. 5' deletion analysis of the OC promoter demonstrated that the promoter (1095 bp) exhibited the greatest response to PTH, whereas the -198 bp construct of the OC promoter, containing only one cAMP response element and OC box, was no longer responsive. The constructs with further deletions (-120, -92, and -74) retained PTH responsiveness, but to a lesser extent. In summary, our results indicate that PTH activation of the OC promoter is a rapid event and mediated by the cAMP-dependent protein kinase A pathway. Although the novel cAMP response region overlapping the OC box is required for activation, full activation may require several cis-acting cAMP response elements or other response elements.
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PMID:Parathyroid hormone (PTH 1-34) regulation of rat osteocalcin gene transcription. 923 53

p53, a tumor suppressor and a transcription factor, has been shown to transcriptionally activate the expression of a number of important genes involved in the regulation of cell growth, DNA damage, angiogenesis, and apoptosis. In a computer search for other potential p53 target genes, we identified a perfect p53 binding site in the promoter of the human type IV collagenase (also called 72-kDa gelatinase or matrix metalloproteinase 2 [MMP-2]) gene. This p53 binding site was found to specifically bind to p53 protein in a gel shift assay. Transcription assays with luciferase reporters driven by the promoter or enhancer of the type IV collagenase gene revealed that (i) activation of the promoter activity is p53 binding site dependent in p53-positive cells but not in p53-negative cells and (ii) wild-type p53, but not p53 mutants commonly found in human cancers, transactivates luciferase expression driven by the type IV collagenase promoter as well as by a p53 site-containing enhancer element in the promoter. Significantly, expression of the endogenous type IV collagenase is also under the control of p53. Treatment of U2-OS cells, a wild-type p53-containing osteogenic sarcoma line, with a common p53 inducer, etoposide, induced p53 DNA binding and transactivation activities in a time-dependent manner. Induction of type IV collagenase expression followed the p53 activation pattern. No induction of type IV collagenase expression can be detected under the same experimental conditions in p53-negative Saos-2 cells. All these in vitro and in vivo assays strongly suggest that the type IV collagenase gene is a p53 target gene and that its expression is subject to p53 regulation. Our finding links p53 to a member of the MMP genes, a family of genes implicated in trophoblast implantation, wound healing, angiogenesis, arthritis, and tumor cell invasion. p53 may regulate these processes by upregulating expression of type IV collagenase.
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PMID:Transcriptional activation by p53 of the human type IV collagenase (gelatinase A or matrix metalloproteinase 2) promoter. 934 94

Knockout of the leukemia inhibitory factor receptor (LIFR) gene results in disrupted placental architecture, imbalanced bone development, and losses of functional neurons. We here report the identification of an enhancer in a functional human LIFR gene promoter and alternative promoter usage by this gene. A single transcription start site was identified in placental JEG-3 cells and a genomic clone containing 4876-nucleotide upstream sequences was found to have promoter activity in JEG-3 cells. However, in osteogenic sarcoma U-2 OS cell, Northern blot using a probe of the first exon detected in JEG-3 cells failed to detect LIFR transcripts. 5'-Rapid amplification of cDNA ends (RACE) revealed an alternative first exon and a 0.6-kilobase pair (kb) 5'-flanking region possessed promoter activity in U-2 OS cells. For the 4.8-kb promoter active in placental cells, a minimal promoter was localized within -162 nucleotides. Three regions increased and one inhibited promoter activity. Subcloning of an activation region (-4876 to -3453 nucleotides) into SV40 promoter either upstream or downstream in either orientation to the luciferase reporter resulted in 10-35-fold luciferase induction, demonstrating the characteristics of an enhancer. Transfections into nine cell lines of different tissue origin indicated that the cloned promoter and enhancer in the 4.8-kb fragment was placental tissue-specific. A 226-base pair fragment (-4625 to -4400 nucleotides) was further localized as the minimal enhancer region, in which deletion of either element A (-4625 to -4581 nucleotides) or element B (-4418 to -4400 nucleotides) resulted in the loss of enhancer activity. Electrophoretic mobility shift assay confirmed that these two elements bind to specific nuclear proteins individually. In the middle region between element A and B, disruption of enhancer integrity also led to a loss of enhancer activity, although two SP1 and three NF-kappaB/c-Rel binding sites did not contribute to enhancer function. These results demonstrate a complex regulation of the human LIFR gene, including alternative promoter usage and tissue-specific elements at the transcription level.
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PMID:Identification of an upstream enhancer within a functional promoter of the human leukemia inhibitory factor receptor gene and its alternative promoter usage. 934 46

Osteocalcin (OC) is a matrix calcium-binding protein expressed in osteoblasts and odontoblasts undergoing mineralization. OC expression is up-regulated in part by signals initiated by basic fibroblast growth factor (FGF2), cyclic AMP or forskolin (FSK), and calcitriol via defined elements and DNA-protein interactions in the OC promoter. We identified the OC gene as a target for transcriptional suppression by Msx2, a homeodomain transcription factor that controls ossification in the developing skull. In this study, we examine the effects of Msx2 expression on OC promoter activation (luciferase reporter) by FGF2/FSK and calcitriol in MC3T3-E1 osteoblasts. Expression of Msx2 decreases basal activity of the 1-kilobase (-1050 to +32) rat OC promoter by 80%; however, the promoter is still inducible 3-fold by calcitriol. By contrast, OC promoter induction by FGF2/FSK is completely abrogated by Msx2. Because intrinsic Msx2 DNA binding activity is not required for the Msx2 suppressor function, we assessed whether Msx2 represses OC activation by regulating DNA-protein interactions at the FGF2 response element (OCFRE) and compared these interactions with those occurring at the calcitriol response element (VDRE). Treatment of MC3T3-E1 cells with FGF2/FSK or calcitriol up-regulates specific DNA-protein interactions at the OCFRE or VDRE, respectively, as detected by gel shift assay. Preincubation of crude nuclear extracts with recombinant glutathione S-transferase (GST)-Msx2 dose-dependently inhibits OCFRE DNA binding activity, whereas GST has no effect. Msx2 itself does not bind the OCFRE. Residues 132-148 required for Msx2 core suppressor function in transfection assays are also required to inhibit OCFRE DNA binding activity. By contrast, GST-Msx2 has no effect on calcitriol-regulated DNA-protein interactions at the VDRE. Using gel shift as an assay, the OCFRE DNA-binding protein OCFREB was purified to about 50% homogeneity from MG63 osteosarcoma cells. Recombinant Msx2 inhibits purified OCFREB DNA binding activity, whereas the Msx2 variant lacking residues 132-148 is inactive. Thus, Msx2 abrogates up-regulation of the OC promoter by FGF2/FSK in part by inhibiting OCFREB binding to the OCFRE.
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PMID:Stimulus-selective inhibition of rat osteocalcin promoter induction and protein-DNA interactions by the homeodomain repressor Msx2. 936 26


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