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

Osteocalcin, the major non-collagenous protein in bone, is transcribed in osteoblasts at the onset of extracellular matrix mineralization. In this study it was demonstrated that sequences located in the first exon of the human osteocalcin gene possess a differentiation-related osteocalcin silencer element (OSE). Osteocalcin was rendered transcribable in UMR-106 cells and proliferating normal osteoblasts after deletion of the -3 to +51 region. Site-specific mutagenesis of this region revealed that a 7 bp sequence (TGGCCCT) (+29 to +35) is critical for silencing function. Mobility shift assays demonstrated that a nuclear factor bound to the OSE. The OSE binding protein was present in proliferating normal pre-osteoblasts and in UMR-106 and ROS 17/2.8 osteosarcoma cells, but was absent from post-proliferative normal osteoblasts. The binding protein was inhibited by fragments containing the +29/+35 sequence, but not by other promoter fragments or by the consensus oligomers of unrelated nuclear factors AP-1 and Sp1. DNase 1 footprinting demonstrated that the OSE binding-protein protected the +17 to +36 portion of the first exon, consistent with the results of mapping studies and competitive mobility shift assays. It is hypothesized that this silencer is activated by complexing of the OSE binding protein to the OSE during the osteoblast proliferation stage and that the OSE binding protein is down-regulated at the onset of extracellular matrix mineralization.
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PMID:Characterization of a silencer element in the first exon of the human osteocalcin gene. 855 66

EB 1089 (1 alpha,25-dihydroxy-22,24-diene-24,26,27-trihomovitamin D3) is a novel, synthetic analog of calcitriol, characterized by two extra double bonds in its side chain. It is less potent than calcitriol in its calcemic action, but is an order of magnitude more potent in its antiproliferative action. The aim of this study was to determine the ability of EB 1089 to induce the well-known biological effects of calcitriol in MG-63 human osteosarcoma cells (i.e. by inhibiting cell proliferation and by induction of differentiation). Both calcitriol and EB 1089 significantly decreased cell growth after 2 days in culture. At 5 days, however, Eb 1089 was more potent than the natural hormone in inhibiting the proliferation of MG-63 cells. Potent effects of EB 1089 on cell differentiation were also seen in the stimulation of alkaline phosphatase activity, cellular vitamin D receptor mRNA levels, and medium osteocalcin synthesis. EB 1089 was clearly more effective than calcitriol in stimulating alkaline phosphatase activity and osteocalcin synthesis. In gel shift assays, the binding of vitamin D receptor to the composite AP-1 plus vitamin-D responsive promoter region of the human osteocalcin gene after EB 1089 treatment was stronger and longer-lasting than after calcitriol treatment.
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PMID:A novel vitamin D analog with two double bonds in its side chain. A potent inducer of osteoblastic cell differentiation. 865 37

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

DNA methylation is a general mechanism of controlling tissue-specific gene expression. Osteocalcin is a bone matrix protein whose expression is limited almost entirely to osteoblasts. We were interested in determining whether the state of methylation of the osteocalcin gene plays a role in its expression by studying human bone-derived (MG-63, U2-Os, SaOs-2) and other types (normal lymphocytes, A-498, Hep G2) of cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that osteocalcin mRNA production is stimulated by 1,25(OH)2D3 in MG-63 and induced in SaOs-2 but not in U2-Os osteoblast-like osteosarcoma cells. Genomic analysis of the human osteocalcin gene showed that the local surroundings of this single-copy gene are identical in all cell lines studied. Using an isoschizomeric pair of restriction enzymes and Southern analysis, we found that the osteocalcin gene is identically methylated in all three osteosarcoma cell lines. The same sites are also methylated in human normal lymphocytes and A-498 kidney cells, whereas the degree of methylation is higher in Hep G2 human hepatocellular carcinoma cells. Furthermore, the osteocalcin gene was identically protected against enzymatic digestion at the chromatin level in normal lymphocytes and in all cell lines studied. Induction of hypomethylation of DNA by 5-azacytidine treatment did not cause an induction of osteocalcin synthesis in these cell lines. On the contrary, it attenuated the induction by 1,25(OH)2D3 in MG-63 cells. In gel mobility shift assays, human vitamin D receptor and the AP-1 transcription factor bound to an unmethylated response element oligonucleotide of the osteocalcin gene with greater affinity than to an in vitro methylated response element. These results indicate that the in vivo methylation state of the osteocalcin gene at sites determined in this study does not correlate with the inducibility of this gene. Nevertheless, the in vitro results clearly indicated that hypomethylation of critical regions of the osteocalcin gene promoter is a potential mechanism influencing effective binding of specific nuclear factors and, consequently, gene expression.
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PMID:State of methylation of the human osteocalcin gene in bone-derived and other types of cells. 925 96

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D, induces nerve growth factor (NGF) synthesis in a variety of different cell lines. The mechanism by which 1,25(OH)2D3 induces NGF, however, is poorly understood. We used a series of full-length and truncated NGF promoter-human growth hormone (hGH) reporter gene plasmids to investigate the mechanism of 1,25(OH)2D3-induced NGF expression in osteoblasts. Untransfected rat osteosarcoma cells (ROS 17/2.8) treated with 1,25(OH)2D3 showed a 2-fold increase in NGF expression compared to control cells. ROS 17/2.8 osteosarcoma cells were transfected with the NGF-hGH reporter plasmids and treated with 10(-)8 M 1,25(OH)2D3. The full-length NGF promoter (-1800 to +120)-hGH reporter construct showed an approximately 2-fold increase in hGH release. Plasmids with successive 5'-deletions showed enhanced hGH expression in treated cells and control cells. A similar series of NGF promoter-hGH reporter gene constructs, lacking the AP-1 site located within the first intron of the NGF gene, were also transiently transfected into ROS 17/2.8 cells. When these cells were treated with the same dose of 1,25(OH)2D3, no increase in hGH expression was seen compared to control cells, demonstrating that this AP-1 site is essential for 1,25(OH)2D3-mediated NGF up-regulation. Since 1,25(OH)2D3 is known to activate the transcription of several genes through its interaction with the vitamin D receptor (VDR), we performed a series of gel electrophoretic mobility shift assays to determine if the VDR binds directly to the AP-1 sequence. No evidence of VDR binding, either as a homodimer or as a heterodimer, to the AP-1 sequence was observed. Treatment of ROS 17/2.8 cells with 1,25(OH)2D3, however, resulted in an increase in AP-1 binding activity; however, no significant changes in c-jun and c-fos levels were observed. Our data show that in osteoblasts, 1,25(OH)2D3 induces NGF expression indirectly by increasing AP-1 binding activity.
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PMID:An AP-1 site in the nerve growth factor promoter is essential for 1, 25-dihydroxyvitamin D3-mediated nerve growth factor expression in osteoblasts. 955 35

The products of c-fos and c-jun proto-oncogenes form the heterodimeric complex AP-1 (activator protein 1), which play an important part in the control of bone cell proliferation and differentiation and in the development of bone tumours. We examined the expression of c-fos and c-jun in a series of 52 primary skeletal neoplasms, using an immunohistochemical method on formalin-fixed, paraffin-embedded sections. The expression of c-fos and c-jun was restricted to bone-forming lesions, while cartilaginous tumours were devoid of immunoreactivity. In benign osteoblastic lesions moderate c-fos and c-jun expression was found in 2 cases (18.1%). The highest levels of c-fos and c-jun expression were detected in high-grade central osteosarcomas (7 of 15 cases with moderate/diffuse expression) while 1 telangiectatic osteosarcoma, 2 low-grade central osteosarcomas, 1 low-grade periosteal osteosarcoma and 7 low-grade parosteal osteosarcomas were either negative or had low expression. The high-grade component of a dedifferentiated parosteal osteosarcoma showed diffuse immunoreactivity for both oncoproteins. Comparison of c-fos and c-jun expression by histological grade showed that high-grade osteosarcomas had a significantly higher expression of both oncoproteins than did low-grade osteosarcomas (P = 0.01, Fisher's exact test). Thus, c-fos and c-jun overexpression may be implicated in the development of high-grade osteosarcomas, but they appear to have little or no relevance for the development of low-grade osteosarcomas and cartilaginous skeletal neoplasms.
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PMID:Immunohistochemical detection of c-fos and c-jun expression in osseous and cartilaginous tumours of the skeleton. 967 92

Human monocyte chemotactic protein-2 (MCP-2) is a member of the CC chemokine family. It is produced by mononuclear leukocytes, diploid fibroblasts, and tumor cells after induction with IL-1beta or IFN-gamma. To understand the transcriptional regulation of the gene, we have analyzed the structure and function of the promoter region. The sequence of the 5'-flanking region was determined and the transcription start site was found to be located at 68 nucleotides upstream of the ATG translation start codon. 5'-Deletion mutants were generated and transfected into E6SM diploid fibroblasts and MG-63 osteosarcoma cells. Expression was measured by luciferase assay in transfected unstimulated cells and after stimulation with IL-1beta, IFN-gamma, or a combination. The region between nucleotides -143 and -73 (relative to the transcription initiation site), containing putative cis-elements for GATA-1, H-APF1, AP-1, and GAS, is important for basal transcription levels in both cell lines. Stimulation for 18 h with IL-1beta alone failed to affect expression of any of the constructs both in diploid fibroblasts and in osteosarcoma cells. In both cell lines IFN-gamma increased the activity of all mutants that possessed the region between -340 and -301. In MG-63 cells, stimulation with the combination of IL-1beta and IFN-gamma caused an additional increase in expression of the constructs from -340 onward. Finally, the presence of transcription factors in nuclear extracts of MG-63 cells and their specificity to bind to various oligonucleotide probes in this [-340; -301] region were evidenced by electromobility shift assays. These results show that IFN-gamma, produced by lymphocytes and NK cells, induces the transcription of the MCP-2 gene in fibroblasts and thereby can indirectly contribute to recruitment of various leukocyte cell types to inflammatory sites.
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PMID:Transcriptional control of the human MCP-2 gene promoter by IFN-gamma and IL-1beta in connective tissue cells. 1049 22

The nuclear phosphoprotein c-Jun is a major component of the AP-1 transcription factor, whose activity is augmented by many oncogenes. An important mechanism to stimulate AP-1 function is N-terminal phosphorylation of c-Jun at the serine residues 63 and 73 by the c-JunN-terminal kinases (JNKs). Mice and cells harboring a mutant allele of c-jun, which has the JNK phosphoacceptor serines changed to alanines (junAA), were used to determine the function of c-Jun N-terminal phosphorylation (JNP) during oncogenic transformation in vitro and in vivo. JunAA immortalized fibroblasts expressing v-ras and v-fos showed reduced tumorigenicity in nude mice, but the efficiency of v-src transformation was unaffected by the lack of JNP. To assess the significance of JNP in tumour development in vivo, two transgenic mouse tumour models were employed. Skin tumour development caused by constitutive activation of the ras pathway by K5-SOS-F expression and c-fos-induced osteosarcoma formation were impaired in mice lacking JNP. Inhibition of JNP may, therefore, be a novel therapeutic strategy to inhibit tumour growth in vivo. Oncogene (2000).
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PMID:Oncogenic transformation by ras and fos is mediated by c-Jun N-terminal phosphorylation. 1085 Oct 65

The expression of MMP13 (collagenase-3), a member of the matrix metalloproteinase family, is increased in vivo as well as in cultured osteosarcoma cell lines by parathyroid hormone (PTH), a major regulator of calcium homeostasis. Binding sites for AP-1 and Cbfa/Runt transcription factors in close proximity have been identified as cis-acting elements in the murine and rat mmp13 promoter required for PTH-induced expression. The cooperative function of these factors in response to PTH in osteoblastic cells suggests a direct interaction between AP-1 and Cbfa/Runt transcription factors. Here, we demonstrate interaction between c-Jun and c-Fos with Cbfa/Runt proteins. This interaction depends on the leucine zipper of c-Jun or c-Fos and the Runt domain of Cbfa/Runt proteins, respectively. Moreover, c-Fos interacts with the C-terminal part of Cbfa1 and Cbfa2, sharing a conserved transcriptional repression domain. In addition to the distal osteoblast-specific element 2 (OSE2) element in the murine and rat mmp13 promoter, we identified a new proximal OSE2 site overlapping with the TRE motif. Both interaction of Cbfa/Runt proteins with AP-1 and the presence of a functional proximal OSE2 site are required for enhanced transcriptional activity of the mmp13 promoter in transient transfected fibroblasts and in PTH-treated osteosarcoma cells.
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PMID:AP-1 and Cbfa/runt physically interact and regulate parathyroid hormone-dependent MMP13 expression in osteoblasts through a new osteoblast-specific element 2/AP-1 composite element. 1127 69

Osteocalcin (OC) is a small (6 kDa) polypeptide whose expression was thought to be limited to mature osteoblasts. The discovery of OC expression in prostate cancer specimens led us to study the regulation of OC gene in androgen-independent metastatic human prostate PC3 cells. An 800-bp human OC (hOC) promoter-luciferase construct exhibited strong basal and vitamin D-induced activity in OC-positive human prostate and osteosarcoma cell lines. Through deletion analysis of the hOC promoter, the functional hierarchy of the cis-acting elements, OSE1, OSE2, and AP-1/VDRE, was established in PC3 cells (OSE1 > AP-1/VDRE > OSE2). By juxtaposing dimers of these 3 cis-elements, we produced a minimal hOC promoter capable of displaying high tissue specific activity in prostate cancer cells. Our study demonstrated three groups of transcription factors, Runx2, JunD/Fra-2, and Sp1, responsible for the high hOC promoter activity in PC3 cells by binding to the OSE2, AP-1/VDRE, and OSE1 elements, respectively. Among the three groups of transcription factors, the expression levels of Runx2 and Fra-2 are higher in the OC-positive PC3 cells and osteoblasts, compared with the OC-negative LNCaP cells. Interestingly, unlike the mouse OC promoter, the OSE1 site in hOC promoter is regulated by members of Sp1 family instead of the osteoblast-specific factor Osf1. The molecular basis for androgen-independent prostate cancer cells behaving like mature osteoblasts may be explained by the interplay and coordination of these transcription factors under the tight regulation of autocrine and paracrine mediators.
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PMID:Regulation of human osteocalcin promoter in hormone-independent human prostate cancer cells. 1168 80


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