Vitamin D-responsive protein-DNA interactions at multiple promoter regulatory elements that contribute to the level of rat osteocalcin gene expression.

Bortell, Rita; Owen, Thomas A.; Bidwell, Joseph P.; Gavazzo, Paola; Breen, Ellen C.; Wijnen, André J. van; DeLuca, Hector F.; Stein, Janet L.; Lian, Jane B.; Stein, Gary S.

doi:10.1073/pnas.89.13.6119

Cited by 65 publications

(45 citation statements)

References 34 publications

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“…Osteocalcin production in MG-63 cells after administration of calcitriol seems to be a good model ing sites and the amount and affinity of these proteins toward their cognate response elements probably determines which one will bind to the AP-1+VDRE region. In addition to the human osteocalcin gene, the promoter region of the rat osteocalcin gene contains multiple vitamin D-responsive regulatory elements which exhibit differences in protein-DNA interactions during cellular development and between normal osteoblasts versus osteosarcoma cells (Bortell et al, 1992. In the human osteocalcin gene, the presence of both AP-1-binding and VDR-binding sites is required for maximal activation of the promoter by calcitriol .…”

Section: Discussionmentioning

confidence: 99%

Functional Interference Between AP‐1 and the Vitamin D Receptor on Osteocalcin Gene Expression in Human Osteosarcoma Cells

Jääskeläinen

Pirskanen

Ryhänen

et al. 1994

European Journal of Biochemistry

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The binding of transcription factor AP-1 and vitamin D receptor (VDR) to the composite AP-1 plus vitamin-D-responsive promoter region (AP-1 +VDRE) of the human osteocalcin gene was characterized in osteocalcin-producing (MG-63) and non-producing (U2-0s, SaOs-2) human osteosarcoma cell lines. In mobility-shift assays with AP-1 +VDRE, AP-1, and VDRE probes and nuclear extracts from these cells, one AP-1-specific and two VDR-specific (fast and slow mobility) interactions were observed. Characterization of the complexes indicated that AP-1 and VDR do not bind simultaneously to the AP-1 + VDRE oligonucleotide. Intensity of the complexes was greatly influenced by cell density: in MG-63 and SaOs-2 cells, AP-1 binding was strong during the proliferative period disappearing at confluency whereas, in U2-0s cells, AP-1 binding was prominent also at the confluent stage. Furthermore, MG-63 cells possessed the faster migrating VDR complex at all stages of confluency whereas, in U2-0s and SaOs-2 cells, it was very weak or absent. There were no detectable differences in the levels of VDR protein between these cell lines. In U2-0s cells, the level of c-jun mRNA was higher than in the other two cell lines, whereas none of these cell lines exhibited detectable levels of c-fos mRNA at the confluent stage. Exogenous c-Jun protein effectively blocked the VDR-DNA interaction. Further, all these cell lines expressed mRNA for retinoid X receptor a (RXRa), the factor suggested to be required for the VDR-DNA interaction. The presence of an accessory factor in the VDR-DNA complexes was indirectly shown by treatment of the cells with 9-cis retinoic acid and by cycloheximide. Both treatments reduced VDR binding without affecting the VDR protein level. These results suggest that AP-1 interferes with VDR binding to the AP-1+VDRE element and that the vitamin D responsiveness of the osteocalcin gene correlates with weak AP-1 binding and strong binding of the faster migrating VDR complex.

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Section: Discussionmentioning

confidence: 99%

Functional Interference Between AP‐1 and the Vitamin D Receptor on Osteocalcin Gene Expression in Human Osteosarcoma Cells

Jääskeläinen

Pirskanen

Ryhänen

et al. 1994

European Journal of Biochemistry

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“…While generally considered a resorptive hormone in bone, it has anabolic effects on the expression of many extracellular matrix proteins that contribute to bone mass. In fact, the majority of the bone/extracellular matrix genes under regulation by 1,25(OH) 2 D 3 are up-regulated by the seco-steroid-including genes encoding alkaline phosphatase (32), osteocalcin (4), and osteopontin (2,3). In this study, we report that the ␣ 1C Ca 2ϩ channel expressed by osteoblastic cells is down-regulated at the mRNA level following exposure to 1 nM 1,25(OH) 2 D 3 for 48 h. This finding is one of a handful of genes negatively regulated by 1,25(OH) 2 D 3 which include aggrecan proteoglycan (33), Id (34), atrial natriuretic peptide (35), and collagen type I (36).…”

Section: Fig 3 Autoradiographs Of Quantitative Rt-pcr Productsmentioning

confidence: 99%

“…1,25-Dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) 1 has long been appreciated as a hormonal modulator of osteoblast function and bone remodeling. 1,25(OH) 2 D 3 , classically considered a resorptive hormone, has the paracrine effects on osteoclasts that are believed to be mediated through separate membrane and nuclear osteoblastic 1,25(OH) 2 D 3 receptor systems (1).…”

mentioning

confidence: 99%

Down-regulation of L-type Ca2+ Channel Transcript Levels by 1,25-Dihyroxyvitamin D3

Meszaros¹,

Karin²,

Akanbi³

et al. 1996

Journal of Biological Chemistry

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Osteoblast Ca2؉ channels play a fundamental role in controlling intracellular and systemic Ca 2؉ homeostasis. A reverse transcription-polymerase chain reaction strategy was used to determine the molecular identity of voltage-sensitive calcium channels present in ROS 17/ 2.8 osteosarcoma cells. The amino acid sequences encoded by the two resultant PCR products matched the The balance between osteoclastic bone resorption and osteoblastic bone formation determines skeletal mass and composition. 1,25-Dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) 1 has long been appreciated as a hormonal modulator of osteoblast function and bone remodeling. 1,25(OH) 2 D 3 , classically considered a resorptive hormone, has the paracrine effects on osteoclasts that are believed to be mediated through separate membrane and nuclear osteoblastic 1,25(OH) 2 D 3 receptor systems (1). Paradoxically, 1,25(OH) 2 D 3 has anabolic effects on osteoblasts, stimulating production of the noncollagenous matrix proteins, osteopontin and osteocalcin (2-4). The exact mechanism by which the bone-forming and -resorbing aspects of the remodeling process are regulated and coupled remains unclear.

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“…This complex transcriptional mechanism involves a specific ligand-receptor interaction, enhanced receptor gene expression, critical receptor phosphorylation sites, formation of multiple receptor-protein complexes, and protein-DNA interactions, as well as overlapping DNA elements at the vitamin D responsive element (VDRE) locus (9)(10)(11)(12)(13)(14)(15). These components of transcriptional control contribute to developmental and physiologic responsiveness of the osteocalcin gene to 1,25(OH)2D3 (16). Transcriptional analysis has established a positive cisacting element, located between nucleotides -465 and -437 of the rat osteocalcin promoter, that confers vitamin D responsiveness (3,17,18).…”

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confidence: 99%

In vivo occupancy of the vitamin D responsive element in the osteocalcin gene supports vitamin D-dependent transcriptional upregulation in intact cells.

Breen

Wijnen

Lian

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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The steroid hormone vitamin D is a principal mediator of skeletal homeostasis. 1,25-Dihydroxyvitamin D3 treatment of ROS 17/2.8 osteoblast-like cells results in a ligand-dependent increase in transcription of the bone-specific osteocalcin gene. This transcriptional upregulation requires the positive cis-acting vitamin D responsive element (VDRE). We have used the ligation-mediated polymerase chain reaction to demonstrate that protein occupancy of the VDRE within the intact cell correlates with increased synthesis of osteocalcin transcripts. These protein-DNA contacts were not present in the absence of vitamin D or in osteosarcoma cells (ROS 24 Rodan (Merck Sharp & Dohme). Cells were maintained in F12 medium containing calcium and charcoal stripped 5% fetal calf serum for 5 days after plating before hormone treatment or vehicle control. 1,25(OH)2D3 was provided by Milan Uskokovic (Hoffman-La Roche).In Vivo Footprint Analysis. In vivo footprinting was performed by exposing cells to 0.1% dimethy sulfate (DMS) (Aldrich) in culture medium for 5 min at room temperature. Methylated genomic DNA was isolated and cleaved with piperidine (Sigma) (26), and the level of methylation protection of G residues was analyzed by the ligation-mediated single-sided polymerase chain reaction (LMPCR) footprinting technique (27). LMPCR was performed with Vent polymerase (New England Biolabs). First-strand synthesis was accomplished with primer 1 (5'-GACAAACTGGCTCCAAC-TCGCATAGC-3'; nt -358 to -383) in first-strand buffer [40 mM NaCl/10 mM Tris HCl, pH 8.9/4 mM MgSO4/0.01% nonacetylated bovine serum albumin (BSA) (New England Biolabs)/0.1% Triton X-100]. The DNA was ligated with the unidirectional linker primer (27). The DNA was amplified in amplification buffer (10 mM NaCl/20 mM Tris HCl, pH 8.9/2 mM MgSO4/0.01% nonacetylated BSA/0.1% Triton X-100) using primer 2 (5'-GCATAGCCTGTGATTTTCAGTGTCT-GCCGT-3'; nt -377 to -406) and the linker oligonucleotide. Eighteen cycles of PCR were performed, each involving denaturation at 96°C, annealing at 56°C, and elongation at 76°C as described (27). Labeling was performed with 32p-end-labeled primer 3 (5'-AGCCTGTGATTTTCAGTGTCTAbbreviations: 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; VDR, vitamin D receptor; VDRE, vitamin D responsive element; DMS, dimethyl sulfate; LMPCR, ligation-mediated single-sided PCR.

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Vitamin D-responsive protein-DNA interactions at multiple promoter regulatory elements that contribute to the level of rat osteocalcin gene expression.

Cited by 65 publications

References 34 publications

Functional Interference Between AP‐1 and the Vitamin D Receptor on Osteocalcin Gene Expression in Human Osteosarcoma Cells

Functional Interference Between AP‐1 and the Vitamin D Receptor on Osteocalcin Gene Expression in Human Osteosarcoma Cells

Down-regulation of L-type Ca2+ Channel Transcript Levels by 1,25-Dihyroxyvitamin D3

In vivo occupancy of the vitamin D responsive element in the osteocalcin gene supports vitamin D-dependent transcriptional upregulation in intact cells.

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