Influence of dexamethasone on the vitamin D–mediated regulation of osteocalcin gene expression

Schepmoes, Grace; Breen, Ellen C.; Owen, Thomas A.; Aronow, Michael S.; Stein, Gary S.; Lian, Jane B.

doi:10.1002/jcb.240470212

Cited by 51 publications

(36 citation statements)

References 44 publications

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“…5D shows that, after phosphatase treatment, there is an increased representation of the more rapidly migrating protein-DNA complex (VDR, lower arrowhead) and several additional high-mobility complexes with a corresponding decreased representation ofthe slower mobility complex (VDR, upper arrowhead). This suggests that phosphorylation of the vitamin D receptor and/or an accessory factor (37-39) is required for formation of the lower-mobility protein-DNA complex and is consistent with reports that phosphorylation of the receptor is required for both ligand binding (40) and for complex formation at the VDRE (41)(42)(43) (44,45) and in transformed osteosarcoma cells (46,47). The synergism observed in osteoblasts could reflect an effect on the state of differentiation (33) or an up-regulation of the vitamin D receptor (44) caused by dexamethasone.…”

Section: Resultssupporting

confidence: 89%

Constitutive transcription of the osteocalcin gene in osteosarcoma cells is reflected by altered protein-DNA interactions at promoter regulatory elements.

Bortell

Owen

Shalhoub

et al. 1993

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

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The bone-specific osteocalcin (OC) gene is transcribed only after completion of proliferation in normal diploid calvarial-derived osteoblasts during extracellular matrix mineralization. In contrast, the OC gene is expressed constitutively in both proliferating and nonproliferating ROS 17/2.8 osteosarcoma cells. To address molecular mechanisms associated with these tumor-related modifications in transcriptional control, we examined sequence-specific interactions of transactivation factors at key basal and hormone-responsive elements in the OC gene promoter. In ROS 17/2.8 cells compared to normal diploid osteoblasts, the absence of a stringent requirement for cessation of proliferation to support both induction of OC transcription and steroid hormonemediated transcriptional modulation is reflected by modifications in transcription factor binding at (s) the two primary basal regulatory elements, the OC box (which contains a CCAAT motif as a central core) and the TATA/glucocorticoid-responsive element domain, and (ii) the vitamin D-responsive element. Particularly striking are two forms of the vitamin D receptor complex that are present in proliferating osteoblasts and osteosarcoma cells. Both forms of the complex are sensitive to vitamin D receptor antibody and retinoic X receptor antibody. After the down-regulation of proliferation, only the lower molecular weight complex is found in normal diploid osteoblasts. Both forms of the complex are present in nonproliferating ROS 17/2.8 cells with increased representation of the complex exhibiting reduced electrophoretic mobility that is phosphorylation-dependent.Genes that support structural and metabolic properties of mature osteoblasts are expressed in actively proliferating transformed osteoblasts and osteosarcoma cells. This is in contrast to the predominant expression ofbone-related genes [e.g., osteocalcin (OC), osteopontin, and alkaline phosphatase] after completion ofproliferation (post-proliferatively) in normal diploid osteoblasts (1,2). It therefore appears that modified growth control in osteosarcoma cells (3) is accompanied by a loss of regulatory mechanisms that mediate the relationships between proliferation and differentiation associated with both progressive development and maintenance of the osteoblast phenotype. This is reflected by loss of the three-stage developmental sequence (4-6) that establishes a bone-tissue-like organization of multilayered cells in a mineralized extracellular matrix (4-10). Thus, in osteosarcoma cells, there is a striking deregulation of growth-related controls whereby proliferation supports initial stages of osteoblast phenotype development, whereas down-regulation of proliferation is required to signal induction of gene expression that is restricted to the post-proliferative period of osteoblast differentiation.To address molecular mechanisms associated with the altered proliferation-differentiation relationship in osteosarcoma cells, we have examined modifications in transcriptional control of cell growth and bone-...

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Section: Resultssupporting

confidence: 89%

Constitutive transcription of the osteocalcin gene in osteosarcoma cells is reflected by altered protein-DNA interactions at promoter regulatory elements.

Bortell

Owen

Shalhoub

et al. 1993

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

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“…3 treatment of ROS 17/2.8 cells with the synthetic glucocorticoid dexamethasone (0.1 ,uM) enhances nuclear protein-DNA interactions in this proximal promoter region (data not shown) further suggests that this segment of the OC gene promoter contains a GRE. These results are consistent with reports in which combined vitamin D and dexamethasone treatment modulates the level of OC expression in a manner different from the effect of either hormone alone (14,19,32).…”

Section: Vitamin D-responsive Protein-dna Interactions In Proximalsupporting

confidence: 92%

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

Bortell

Owen

Bidwell

et al. 1992

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

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The observation that vitamin D-mediated enhancement of osteocalcin (OC) gene expression is dependent on and reciprocally related to the level of basal gene expression suggests that an interaction of the vitamin D responsive element (VDRE) with basal regulatory elements of the OC gene promoter contributes to both basal and vitamin D-enhanced transcription. Protein-DNA interactions at the VDRE of the rat OC gene (nudeotides -466 to -437) are reflected by direct sequence-specific and antibody-sensitive binding of the endogenous vitamin D receptor present in ROS 17/2.8 osteosarcoma nuclear protein extracts. In addition, a vitamin D-responsive increase in OC gene transcription is accompanied by enhanced non-vitamin D receptor-mediated protein-DNA interactions in the "TATA" box region (nucleotides -44 to +23), which also contains a potential glucocorticoid responsive element. Evidence for proximity of the VDRE with the basal regulatory elements is provided by two features of nuclear architecture. (a)Nuclear matrix attachment elements in the rat OC gene promoter that bind nuclear matrix proteins with sequence specificity may impose structural constraints on promoter conformation. (ii) Limited micrococcal nuclease digestion and Southern blot analysis indicate that three nucleosomes can be accommodated in the sequence spanning the OC gene VDRE, the OC/CCAAT box (nucleotides -99 to -76), and the TATA/glucocorticoid responsive element, and thereby the potential distance between the VDRE and the basal regulatory elements can be reduced. A model is presented for the contribution of both the VDRE and proximal promoter elements to the enhancement of OC gene transcription in response to vitamin D. The vitamin D receptor plus accessory proteins may function cooperatively with basal regulatory factors to modulate the extent to which the OC gene is transcribed.Osteocalcin (OC) is a bone-specific protein that is expressed with the onset of mineralization of the collagenous bone extracellular matrix in vivo (1) and in vitro (2, 3). Normal diploid osteoblasts express the OC gene after proliferation has ceased during development of the bone tissue-like extracellular matrix, as reflected by transcription, cellular mRNA accumulation, and OC protein biosynthesis. In contrast, OC gene expression is constitutive in ROS 17/2.8 osteosarcoma cells during and after proliferation (4). In both normal diploid and transformed bone cells, vitamin D enhances OC gene expression at the three principal levels-transcription, mRNA accumulation, and protein synthesis (5, 6).OC gene transcription is controlled by the combined activity of a series of positive and negative promoter elements that respond to physiological mediators. Basal and enhanced transcription represent key components of control that are regulated by sequence-specific protein-DNA interactions. Consequently, we examined two proximal basal promoter elements, the OC box and "TATA" box, as well as the vitamin D responsive element (VDRE). The OC box [nucleotides (nt) -99 to -76], whic...

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“…Increased osteocalcin has been linked to increased turnover in rats after Ovx as well (41,42). Whether the increased osteocalcin in PTHtreated groups is the result of increased 1,25 (OH)2D cannot be determined but 1,25(OH)2D has been shown to stimulate osteocalcin expression in vitro (43).…”

Section: Discussionmentioning

confidence: 99%

Loss of cancellous bone mass and connectivity in ovariectomized rats can be restored by combined treatment with parathyroid hormone and estradiol.

Shen¹,

Dempster²,

Birchman³

et al. 1993

J. Clin. Invest.

172

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To evaluate the potential use of a combination of antiresorption and bone formation-promoting agents as a treatment for postmenopausal osteoporosis, we examined the effects of combined and separate administration of estrogen ( 17,8-estradiol, 30 ;Lg/ kg per d, s.c.) and parathyroid hormone (rPTH 40 ;&g/ kg per d, s.c.) on the proximal tibia of ovariectomized (Ovx) rats. The treatments lasted for 4 wk and were initiated 1, 3, and 5 wk after surgery. Ovx resulted in rapid loss of cancellous bone volume (Cn-BV/TV) as well as trabecular connectivity, as determined by two dimensional strut analysis. When administered in a preventive mode, treatment beginning I wk postOvx, estrogen or PTH treatment alone preserved Cn-BV/TV and trabecular connectivity, and combined estrogen and PTH treatment caused a 40% increment in Cn-BV/TV while maintaining comparable trabecular connectivity with that seen in the Sham-operated animals. When administered in a curative mode to rats with established osteoporosis, treatments beginning 3 or 5 wk post-Ovx, estrogen or PTH treatment alone prevented further loss of connectivity and Cn-BV/TV, whereas the combined treatment resulted in as much as a 300% improvement in one of the parameters of trabecular connectivity, node to node strut length, and a 106% increase in Cn-BV/TV, with respect to the bone status at the initiation of treatment. The beneficial effects of this combined treatment derive from estrogen's ability to prevent accelerated bone resorption and, simultaneously, PTH's promotion of bone formation. These data demonstrate, in an animal model, that therapies can be devised to cure the skeletal defects associated with established osteoporosis. (J.

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Influence of dexamethasone on the vitamin D–mediated regulation of osteocalcin gene expression

Cited by 51 publications

References 44 publications

Constitutive transcription of the osteocalcin gene in osteosarcoma cells is reflected by altered protein-DNA interactions at promoter regulatory elements.

Constitutive transcription of the osteocalcin gene in osteosarcoma cells is reflected by altered protein-DNA interactions at promoter regulatory elements.

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

Loss of cancellous bone mass and connectivity in ovariectomized rats can be restored by combined treatment with parathyroid hormone and estradiol.

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