Contributions of distal and proximal promoter elements to glucocorticoid regulation of osteocalcin gene transcription.

Abstract

Previous studies identified several glucocorticoid response elements (GREs) in the 5'-promoter region of the rat osteocalcin (OC) gene by purified receptor binding. The present study addresses functionality of the GRE sequences in the proximal promoter at nucleotide (nt) -16 to -1 downstream of the TATA element together with the GRE half-element in the OC box at nt -86 to -81. This was done by assaying glucocorticoid responsiveness [at 10(-6) M dexamethasone (DEX)], and in combination with 10(-8) M 1,25-dihydroxyvitamin D3, of a series of deleted and mutated OC promoter reporter constructs (OCCAT) in osteoblast-like cells, the ROS 17/2.8 rat osteosarcoma line. Promoter deletion analysis revealed an additional GRE in the distal promoter at nt -697 to -683 that functions to suppress OC transcription. In the absence of this upstream negative GRE (nGRE), the -531 OCCAT construct exhibited enhanced promoter activity in response to DEX (1.8-fold DEX/Control), but further deletion (-348 and -108 OCCAT constructs) restored DEX suppression to OC promoter activity (0.6- and 0.8-fold DEX/Control, respectively). Mutations introduced in both the proximal GRE (nt -16 to -1) and the half-GRE in the OC box, or in the proximal GRE alone, nearly abrogated DEX responsiveness of OC promoter activity. Both distal and proximal GREs specifically bound glucocorticoid receptor present in ROS 17/2.8 nuclear extracts as shown by competition with wild type and mutated oligonucleotides and antibody inhibition of binding. Furthermore, both GREs, independently, conferred DEX-responsive transcriptional repression to the heterologous thymidine kinase basal promoter. We also report that glucocorticoid suppression of 1,25-dihydroxyvitamin D3-stimulated transcription occurs independently of distal or proximal GREs. Taken together, these results demonstrate that in vivo responsiveness of OC to DEX involves the integrative activities of several functional promoter elements.

YY1 regulates vitamin D receptor/retinoid X receptor mediated transactivation of the vitamin D responsive osteocalcin gene

“…The OC gene promoter contains multiple positive and negative regulatory elements, including a VDRE (14)(15)(16)(17)(18)(19). The VDRE plays a key role in the transcriptional regulation of OC gene expression in osteoblasts both in vivo and in vitro (20)(21)(22)(23).…”

Cbfa1 Isoforms Exert Functional Differences in Osteoblast Differentiation

“…In the transcriptional activation of the OSC gene, the discrepancy between the results of the promoter analysis and those of the stable transfection experiment may be due to the region of the construct we used in luciferase assay, i.e. endogenous OSC gene expression is regulated by many factors and many functional elements in the gene, such as CREB/CRE (44), MSX (45), and GR/GRE (46). In the transcriptional activation of the OPN gene, the results of the promoter analysis and those of the stable transfection experiment correlated very well; this reflected that the important regulatory element in osteoblastic lineage cells exists in the regions of our construct (38,47).…”

Multiple levels of steroid hormone‐dependent control of osteocalcin during osteoblast differentiation: Glucocorticoid regulation of basal and vitamin D stimulated gene expression

“…We experimentally addressed a paradox. Dex inhibits basal and vitamin D-stimulated OC mRNA expression through specific gene regulatory sequences of both the rat and human OC genes in ROS 17/2.8 cells [Schepmoes et al, 1991;Bortell et al, 1992;Morrison and Eisman, 1993;Morrison et al, 1989;Aslam et al, 1995;Heinrichs et al, 1993] and human osteoblasts [Subramaniam et al, 1992;Wong et al, 1990]. However, Dex promotes OC expression when acting as a potent differentiation inducing agent for marrow cells [Kasugai et al, 1991;Leboy et al, 1991;Cheng et al, 1996] or fetal rat calvarial derived osteoprogenitor cells Shalhoub et al, 1992].…”

“…The rat and human OC promoters contain multiple glucocorticoid responsive elements (GREs) [Stromstedt et al, 1991;Morrison and Eisman, 1993;Morrison et al, 1989;Aslam et al, 1995;Heinrichs et al, 1993]. In the rat gene, GREs in the distal (nt Ϫ697 to Ϫ683) and proximal promoter regions (nt Ϫ16 to Ϫ1 nt) bind purified glucocorticoid receptor and have been shown to modulate negatively the 1,25(OH) 2 D 3 -induced transcription of osteocalcin in ROS 17/2.8 osteosarcoma cells [Aslam et al, 1995]. However, in normal diploid osteoblasts, the mechanisms by which OC mRNA and protein synthesis are increased during glucocorticoid treatment are unclear.…”