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 thr ough interactions with a specific intracellular receptor (glucocortico id receptor) that regulates gene transcription through a cognate eleme nt, the glucocorticoid response element (GRE), in the promoter of targ et genes. In bone formation glucocorticoids stimulate osteoblast diffe rentiation and the formation of bone matrix. Recent studies have demon strated that the induction of the bone sialoprotein (BSP) gene is asso ciated with osteoblast differentiation and de novo bone formation. To determine the molecular pathways of glucocorticoid regulation of BSP e xpression, we have analyzed the effects of the synthetic glucocorticoi d, dexamethasone, on the expression of the BSP by bone cells in vitro. At 10 nM, dexamethasone induced BSP expression in association with bo ne tissue formation by confluent fetal rat calvarial cells and adult r at marrow cells and also stimulated BSP expression up to sixfold in os teoblastic cells (UMR 106-6 and ROS 17/2.8 cells). Most of the stimula tion was blocked by cycloheximide, indicating direct and indirect mech anisms 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 -beta-D-ribofuanosyl benzimidazole) by Northern hybridization revealed that the stability of the BSP mRNA was not significantly altered by d examethasone, 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 downs tream to +2282 in the first intron. Transient transfection analyses, u sing various rat BSP promoter constructs linked to a luciferase report er gene, and gel mobility shift assays were used to identify a putativ e glucocorticoid response unit comprising three GRE half-sites and a p utative AP-1 site, located within positions -906 to -931 upstream from the translation start site of the BSP gene promoter. BSP transcriptio n was stimulated approximate to 1.5-fold by dexamethasone through this GRE, indicating that its direct effects are mediated by glucocorticoi d receptor binding to this site. These studies, therefore, have identi fied both indirect and direct pathways of glucocorticoid regulation of BSP gene expression, the direct effects being mediated by a GRE in th e rat BSP promoter through which the effects of glucocorticoids on BSP gene transcription appear to be regulated.