Bone Sialoprotein Promoter Research Articles

Glucocorticoid Regulation of Bone Sialoprotein (BSP) Gene Expression. Identification of a Glucocorticoid Response Element in the Bone Sialoprotein Gene Promoter

Glucocorticoids modulate the development and growth of many organs through interactions with a specific intracellular receptor (glucocorticoid receptor) that regulates gene transcription through a cognate element, the glucocorticoid response element (GRE), in the promoter of target genes. In bone formation glucocorticoids stimulate osteoblast differentiation and the formation of bone matrix. Recent studies have demonstrated that the induction of the bone sialoprotein (BSP) gene is associated with osteoblast differentiation and de novo bone formation. To determine the molecular pathways of glucocorticoid regulation of BSP expression, we have analyzed the effects of the synthetic glucocorticoid, dexamethasone, on the expression of the BSP by bone cells in vitro. At 10 nM, dexamethasone induced BSP expression in association with bone tissue formation by confluent fetal rat calvarial cells and adult rat marrow cells and also stimulated BSP expression up to sixfold in osteoblastic cells (UMR 106–6 and ROS 17/2.8 cells). Most of the stimulation was blocked by cycloheximide, indicating direct and indirect mechanisms of BSP gene regulation. Nuclear ‘run-on’ transcription analysis revealed an up to twofold increase in transcription corresponding to the increase in mRNA that was unaffected by cycloheximide. Analysis of BSP mRNA in the presence of a transcription inhibitor (5,6–dichloro-1-β-d-ribofuanosyl benzimidazole) by Northern hybridization revealed that the stability of the BSP mRNA was not significantly altered by dexamethasone, indicating that the major, indirect, stimulation of BSP expression involves a nuclear post-transcriptional mechanism. To study the direct effects of dexamethasone, nucleotide sequence analysis of the rat BSP promoter was extended upstream to position−2992 and downstream to +2282 in the first intron. Transient transfection analyses, using various rat BSP promoter constructs linked to a luciferase reporter gene, and gel mobility shift assays were used to identify a putative glucocorticoid response unit comprising three GRE half-sites and a putative AP-1 site, located within positions –906 to –931 upstream from the translation start site of the BSP gene promoter. BSP transcription was stimulated ≈1.5-fold by dexamethasone through this GRE, indicating that its direct effects are mediated by glucocorticoid receptor binding to this site. These studies, therefore, have identified both indirect and direct pathways of glucocorticoid regulation of BSP gene expression, the direct effects being mediated by a GRE in the rat BSP promoter through which the effects of glucocorticoids on BSP gene transcription appear to be regulated.

Characterization of the Human Bone SialoProtein (BSP) Gene and its Promoter Sequence

Bone sialoprotein (BSP) is a major structural protein of the bone matrix that is specifically expressed by fully-differentiated osteoblasts. To characterize the gene and to study the tissue-and differentiation stage-specific regulation of BSP gene transcription we have isolated and partially sequenced two overlapping genomic fragments which span the complete human BSP gene and its promoter region. The approximately 15 kb gene comprises seven exons of 82 bp, 68 bp, 51 bp, 78 bp, 63 bp, 159 bp and 2.5 kb (1-7, respectively), separated by six introns of approximately 3 kb, 92 bp, 95 bp, approximately 3 kb, approximately 0.5 kb and approximately 4.5 kb. All of the intron-exon boundaries defining the splice sites conform to the consensus sequence of: AG at the 3' splice site; and GT at the 5' splice site, except the 3' splice site of exon 1. The first exon encodes the 5'-UTR, the second exon the signal sequence and the first two amino acids, exons 3 and 4 the Tyr-and Phe-rich amino terminus, and exon 5 the first segment of polyglutamic acid. Exon 7 encodes over half of the protein including a second polyglutamic acid segment, the RGD cell attachment motif, the sulphated tyrosine-rich C-terminus and the 3'-UTR. The promoter region is characterized by an inverted TATA-like sequence (TTTATA), nts -28 to -23 from the transcriptional start site (+1), and an inverted CCAAT box (ATTGG) at -54 to -50. Analysis of chimeric constructs fused to a CAT reporter gene indicate that the presence of both the inverted TATA-like sequence and CCAAT elements are required for basal promoter activity. Comparison of the human BSP promoter with the rat BSP promoter (Li and Sodek, 1993) reveal that the nature and position of the inverted TATA-like sequence and CCAAT box together with an Ap1 (-148 to -142), CRE (-122 to -116) and a homeobox-binding site (-200 to -191) have been conserved. A putative Glucocorticoid Response Unit (GRU) consisting of a Glucocorticoid Response Element (GRE) and an overlapping direct repeat (DR5) similar to the retinoic acid receptor element (RARE) is present at -1038 to -1022. These studies have defined the structure of the human BSP gene and have identified novel transcriptional elements in the promoter that may be involved in the developmentally regulated, tissue specific expression of this gene.