Identification of multiple glucocorticoid receptor binding sites in the rat osteocalcin gene promoter

Abstract

The biosynthesis of osteocalcin (OC), a bone-specific, noncollagenous protein, is stringently regulated during differentiation of the osteoblast phenotype. Glucocorticoids, and also 1,25(OH)2D3, mediate the developmental regulation of OC gene transcription. In this study, we established that the -1097 to +23 promoter (pOCZCat) of the rat OC gene confers glucocorticoid responsiveness to both basal and vitamin D-induced OC expression. The presence of multiple glucocorticoid receptor (GR) binding sites in the proximal rat OC gene promoter was determined by the combined use of DNase I footprinting, dimethyl sulfate fingerprinting, and gel mobility shift analysis with glucocorticoid receptor protein. One glucocorticoid receptor binding element (GRE) resides immediately downstream of the TATA box (-16 to -1). In vivo activity was established by cotransfection of ROS 17/2.8 osteosarcoma cells with an OC-CAT construct in the presence of cloned GRE sequences (wild type or mutant) as competitors. A putative second, less protected GR binding site is located further upstream in the OC gene basal promoter within the region overlapping the TATA box. This is in direct contrast to the organization of GREs in the human OC proximal promoter wherein GR binding at the upstream GRE overlapping the TATA is stronger than at the downstream GRE. In addition, we detected sequence-specific binding of GR protein to another basal promoter element, the OC box (-99 to -76), which contains a central CCAAT motif. The presence of multiple GR binding sites in the rat OC gene proximal promoter indicates that regulation of basal and vitamin D-enhanced transcription by glucocorticoids may involve the integrated activities of multiple, independent GREs.

New mechanisms of glucocorticoid-induced insulin resistance: make no bones about it

“…These take advantage of the fact that many of the genes associated with side effects (e.g., osteocalcin; ref. 17) are modulated through glucocorticoid receptor binding to glucocorticoid response elements, whereas many of the therapeutic effects occur through interactions with glucocorticoid receptor-associated transcription factors, such as AP1 (22). A number of selective glucocorticoid receptor modulators have been studied (22), and at least one is currently undergoing a phase II clinical trial for the treatment of rheumatoid arthritis (NCT01393639).…”

“…The observation that glucocorticoids inhibit the transcriptional activation of osteocalcin through transrepression made decreases in osteocalcin a possible candidate for glucocorticoid-mediated bone loss (17). However, knockout of the osteocalcin gene in mice results in increased, not decreased, bone formation (18).…”

Glucocorticoids inhibit osteocalcin transcription in osteoblasts by suppressing Egr2/Krox20‐binding enhancer

“…Early studies suggested that direct binding of the glucocorticoid receptor (GR) at the vicinity of the OC gene TATA box contributed to transcriptional repression (16,(25)(26)(27). Other GR binding sites were found at the OC box (28,29) and at promoter domains that were more distal (28). However, repression by competition at basal cis-acting regulatory elements is unlikely shared by the as-yet-unidentified glucocorticoidinhibited osteoblastic genes for which OC serves as a model.…”

The Factor Binding to the Glucocorticoid Modulatory Element of the Tyrosine Aminotransferase Gene Is a Novel and Ubiquitous Heteromeric Complex

“…Under most circumstances, more than one protein would be required to cover such a large stretch of DNA. When compared to molecular weight markers in the gel shift assay (41), the size of the GMEB was calculated to be ϳ550 kDa; the sizes of the CREB-containing bands were about 310 and 360 kDa (data not shown). A similar very large size of 600 kDa for the GMEB was observed by gel shift assays of the peak binding activity after fractionation by size exclusion chromatography on Superose 6 HR (Fig.…”