RU486 is also known as mifepristone, and has a systematic International Union of Pure and Applied Chemistry (IUPAC) name of 11β-[p-(Dimethylamino)phenyl]-17β- hydroxy-17-(1-propynyl)estra-4,9-dien-3-one. Cortisol is also known as hydrocortisone and has the IUPAC name of (11β)-11,17,21-trihydroxypregn-4-ene-3,20-dione. Corticosterone has the IUPAC name of (11β)-11,21-dihydroxypregn-4-ene-3,20-dione. 1,25-dihydroxy-vitamin D₃, (abbreviated in this paper as 1,25(OH)₂D₃) is also known as 1a,25 dihydroxyvitamin D₃, 1a,25-Dihydroxycholecalciferol and calcitriol, and has the IUPAC name of (1R,3S)-5-[2-[(1R,3aR,7aS)-1-[(2R)-6-hydroxy-6-methyl-hetan-2-yl]-7a-methyl-2,3,3a,5,6,7-hexahydro-1H-inden-4-ylidene]ethylidene]-4-methylidene-cyclohexane-1,3-diol. Dexamethasone, also known as prednisolone F, has the IUPAC name of (8S,9R,10S,11S,13S,14S,16R,17R)-9- Fluoro-11,17-dihydroxy-17-(2-hydroxya-cetyl)-10,13,16- trimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one.

RU486 was obtained from Roussel-UCLAF (France). Cortisol, corticosterone, dexamethasone and 1,25(OH)₂D₃ were obtained from Sigma-Aldrich. Hormones were stored under nitrogen in ethanol at −20˚C in the dark and diluted appropriately prior to use.

Rat osteoblastic sarcoma cells ROS 17/2.8 were cultured using Ham’s F12 medium or Dulbecco’s modification of Eagle’s medium (DMEM) (Flow Laboratories) supplemented with 10% fetal calf serum (FCS) in a 5% carbon dioxide atmosphere at 37˚C. The plasmid construct pOSCAT2 [7] contains the promoter of the human osteocalcin gene driving the chloramphenicol acetyl transferase (CAT) gene. pSV2NEO contains the neomycin phosphotransferase gene driven by the SV40 virus major late promoter. pOSCAT2 and pSV2NEO were mixed equimolar and precipitated with calcium phosphate according to Gorman et al. [14]. Fifty mg of precipitated DNA was added per flask to five 150 cm² flasks of ROS17/2.8 cells at 2 × 10⁶ cells per flask, grown in DMEM augmented with 10% FCS. After six hours the cells were shocked with 10% glycerol in DMEM (plus 10% FCS) for one minute and the original medium replaced. Cells were left undisturbed for three days before the addition of antibiotic G418 (Life technologies) at 500 mg/ml, a concentration established to be toxic to ROS17/2.8 cells. Cells were mixed and re-plated after six days into Ham’s F12 medium (plus 10% FCS) supplemented with ROS17/2.8 conditioned medium (50%), containing G418. Cells were re-fed conditioned selective medium every three days to remove dead cells and replated into smaller flasks at weekly intervals. After three weeks, a pool of G418 resistant cells had developed which had a high plating efficiency in the presence of G418. These cells have a high level of CAT activity that was inducible by 1,25(OH)₂D₃ and were used in hormone treatment experiments. In the pool of selected cells, G418 selection was not necessary to maintain either G418 resistance or CAT activity, which has remained constant for over a year in the absence of G418 selection.

Transfected cells were expanded in Hams-F12 medium (plus 10% FCS) in 150 cm² flasks. Two days prior to harvesting for hormone treatments, medium was replaced with DMEM supplemented with 2% charcoal stripped FCS (CS-FCS, Life technologies). Cells were harvested, washed and re-plated into six well plates at a density of 2.5 × 10⁵ cells per well in 2.5 ml of DMEM (plus 2% CS-FCS) medium. Hormone treatments were compared to appropriate controls of the vehicle (ethanol) at the same concentration. 2.5 ml of hormone concentrate or vehicle was added to the well after the cells had plated down. Cells were incubated at 37˚C in 5% CO₂ for 36 hours prior to harvest.

CAT activity assays were based on the transfer of carbon-14 labeled acetyl groups from ¹⁴C acetyl-CoA (Perkin Elmer) to unlabelled chloramphenicol [15]. Cells were harvested by trypsinization, recovered in DMEM (plus 10% FCS) medium, washed in phosphate buffered saline and lysed by three cycles of freeze-thaw for CAT assay in a volume of 100 ml of which 10 ml was used to assay CAT activity for one hour at 37˚C in a final reaction volume of 100 ml. Three sequential organic extractions in 100 ml ice-cold ethyl acetate were used to separate labeled chloramphenicol from ¹⁴C acetyl-CoA. Prior to counting, an aqueous wash of 100 ml phosphate buffered saline was done and the organic phase carefully separated and counted in scintillation fluid using the carbon- 14 channel of a Packard liquid scintillation counter. All hormone treatments were for 36 hours (in triplicate) and data is expressed as the mean ± standard error of the mean of disintegrations per minute (dpm) of radioactive acetyl groups transferred from ¹⁴C acetyl-CoA to chloramphenicol per hour per 10⁶ cells.

The web based transcription element search system (TESS) computer program [16] was used to search for motifs related to glucocorticoid receptor binding sites. In order to identify nGRE similar to those described by Surjit et al. [13] the simple motifs CTCC and GAGG were used.

Dexamethasone is a powerful synthetic glucocorticoid known to repress vitamin D induction of human osteocalcin promoter activity [7]. Cortisol is a natural glucocorticoid and the primary circulating glucocorticoid in humans, while corticosterone is the primary physiological glucocorticoid in rodents. These three agents were compared with RU486 in order to establish if basal, un-induced osteocalcin promoter activity could be affected within a short (36 hours) treatment window. Transfected cells were treated with increasing concentrations of these agents (Figure 1) and CAT activity measured as a surrogate for promoter activity. The three glucocorticoids resulted in sensitive repression of osteocalcin basal promoter activity in a manner consistent with dexamethasone having a higher affinity for the GR than cortisol or corticosterone. Despite the differences in the concentration dependence of the repression of basal activity, all three steroids attained the same extent of suppression at maximal concentrations, reducing osteocalcin basal promoter activity to about 40% of the vehicle treated control. The anti-hormone RU486, which might have been expected to have no effect, had a suppressive effect as well, that was detected at reasonably low concentrations (10^–8 and 10^{–9 M). In contrast to dexamethasone and cortisol, the repression effect of RU486 on the osteocalcin promoter did not attain the same magnitude as the other authentic agonists (Figure 1).}

Considering that cortisol represses osteocalcin promoter basal activity in transfected cells, the effect of cortisol on

1,25(OH)₂D₃ induction was investigated. Dose response curves of 1,25(OH)₂D₃ mediated induction (with concentrations from 10⁻¹² to 10⁻⁸ M) were established at increasing cortisol concentrations with hormones added simultaneously. The control experiment of 1,25(OH)₂D₃ induction (Figure 2) shows around five-fold induction within 36 hours of treatment with 1,25(OH)₂D₃ and a saturated induction at 10⁻⁹ M, with a half maximal dose of between 10^–11 and 10^{–10 M (Figure 2). Increasing concentrations of cortisol have a profound repressive effect on 1,25(OH)}₂D₃ induction, in keeping with represssion of basal activity seen already (Figure 1). Cortisol at 10^{–8 M results in an effective halving of the 1,25(OH)}₂D₃ mediated promoter induction, meaning that physiologically sensible levels of cortisol within a short time frame could suppress 1,25(OH)₂D₃ mediated promoter induction. Furthermore, cortisol at 10^–7 and 10^{–6 M} results in virtual shut down of vitamin D effects on the osteocalcin promoter. At the lower concentrations of 1,25(OH)₂D₃, the repression overcomes induction to such an extent that the level of CAT activity is reduced below the control. Therefore, in the presence of 10^{–7 M cortisol a concentration of 1,25(OH)}₂D₃ of 10^{–10 M is required merely to compensate the gene promoter activity to the normal level.}

RU486 had a repressive effect on osteocalcin promoter basal activity: therefore the action of RU486 on cortisolmediated repression of vitamin D induction could not be

predicted. Although RU486 exhibited weak agonist activity on osteocalcin basal activity (Figure 1), there was virtually no repression of 1,25(OH)₂D₃ induced CAT activity by RU486 at 10^{–8 M and 10–7 M concentrations and only a minor effect (activity at 91%) when used at 10–6 M, in marked contrast to dexamethasone and cortisol (Figure 3). Multiple dose response curves of the interacttion between RU486, cortisol and 1,25(OH)}₂D₃ were established (Figure 4) at the following concentrations: 1,25(OH)₂D₃ was at 10^–10 and 10^{–9 M; RU486 varied from 10–10 M to 10–6 M; cortisol varied from 10–10 M to 10–6 M. As expected, low cortisol concentrations (10–9 M), resulted in weak repression of 1,25(OH)}₂D₃ induction (Figure 4(a)): surprisingly addition of a small amount of RU486 (compare 10^–10 to 10^{–9 M RU486 at both concentrations of 1,25(OH)}₂D₃ resulted in slightly more inhibition. As RU486 concentration was increased, however, repression by cortisol was relieved. At higher concentrations of cortisol (10^{–8 M, Figure 4(b)) the reversal of cortisol repression mediated by RU486 is more obvious, since repression was a more potent. Increasing RU486 results in dose-dependent steady increase in CAT activity, due to reversal of cortisol repression of 1,25(OH)}₂D₃ induction (Figure 4(b)). However, it is notable that the relief of repression curve alters direction at 10^{–6 M RU486, becoming inhibitory again (Figure 4(b)). This suggests that lower concentrations of RU486 act as a pure antagonist while very high levels of RU486 may act as a GR agonist. At the highest concentrations of cortisol tested in this experiment (10–7 M, Figure 4(c)), RU486 shows a sigmoidal curve of relief of cortisol repression with apparent saturation of the effect at 10–7} to 10^{–6 M RU846. However, a notable feature of this graph (Figure 4(c)) is that RU486 does not compensate the osteocalcin promoter activity back to 100% activity: the}

level expected for complete reversal of cortisol repression. Rather, the osteocalcin promoter activity in the presence of RU486 at 10^{–6 M, cortisol at 10–7 M and 1,25(OH)}₂D₃ at 10^{–9 M does not exceed approximately 75% of the activity of 1,25(OH)}₂D₃ alone. Although speculative, the inability of RU486 to totally relieve the repression effected by cortisol at 10^{–7 M may indicate that the system is limited to a new set point.}

The osteocalcin promoter sequence was searched for a classical GRE (motif shown in Figure 5(a)) and recently defined active nGRE sequences (based on the motif CTCC or GGAG, Figure 5(b)). Simple search motifs were used search the construct sequence for both canonical GRE and recently described active nGRE aided a transcription element search system (TESS) computer program [16]. A total of 31 perfect CTCC and GAGG motifs were identified in the osteocalcin promoter sequence present in the construct. None of these were arranged where half-sites were in proximity as inverted repeats as described by Surjit et al. [13]. Of these, a single half site exists within a sequence adjacent to a defined RUNX2 binding site. Sequences that matched consensus glucocorticoid or progesterone receptor binding sites (GRE/PRE) were found using TESS (Figure 5), including the sequence overlapping the TATA box that matched prior data [7,12]. The vitamin D response element (VDRE) did not overlap or contain candidate nGRE motifs (Figure 5(b)).