Transcriptional Synergism between Vitamin D-responsive Elements in the Rat 25-Hydroxyvitamin D3 24-Hydroxylase (CYP24) Promoter

Abstract

The hormone 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 1 or calcitriol) is a pleiotropic secosteroid that functions in the regulation of calcium homeostasis, cellular differentiation and proliferation, and immune function (1-5). Nuclear actions of 1,25-(OH) 2 D 3 involve the transcriptional regulation of gene expression, which is mediated by the vitamin D receptor (VDR), a ligand-activated transcription factor that belongs to the nuclear receptor superfamily (1, 6 -10). Activated VDR can bind as either a homodimer or a heterodimeric complex to a DNA sequence known as the vitamin D-responsive element (VDRE) present in the promoter of target genes (11-13). Heterodimers consisting of VDR and retinoid X receptor (RXR) are widely documented (12), although VDR heterodimeric complexes have also been demonstrated for both the retinoic acid receptor (14, 15) and the thyroid hormone receptor (16). Vitamin D-responsive elements generally display a binding motif that consists of two imperfect, direct repeat hexameric sequences (i.e. halfsites) that are separated by 3 bp or, more rarely, by 6 bp; these VDREs are referred to as DR-3 and DR-6, respectively (12).Metabolic inactivation of 1,25-(OH) 2 D 3 and conversion to water-soluble calcitroic acid occurs through the C-24 oxidation pathway. The initial step in this pathway involves the 24-hydroxylation of 1,25-(OH) 2 D 3 by the mitochondrial enzyme 25-hydroxyvitamin D 3 24-hydroxylase (CYP24) (17, 18). Rats fed a normal diet express a low level of CYP24, predominantly in the kidney. However, the enzyme can be substantially induced in kidney and intestine (19 -23) and various other cells (24 -29) by 1,25-(OH) 2 D 3 treatment. Up-regulation of CYP24 expression (19 -23) increases the metabolic clearance of 1,25-(OH) 2 D 3 , and, thereby, feedback regulates the hormone's ambient and cellular levels (17, 18). The mechanism whereby 1,25-(OH) 2 D 3 acts to modulate cellular CYP24 expression is of fundamental importance to understanding the hormone's role in health and disease.Molecular regulatory studies of CYP24 gene expression by 1,25-(OH) 2 D 3 are in progress and promoter analysis data for rat (14, 30 -33) and human (34) have been reported. In the rat CYP24 gene promoter, three VDREs on the antisense strand have been identified. We have previously defined the proximal VDRE (30) in its native promoter context, while the two more distal VDREs have been tested by fusing to a heterologous promoter (14, 31). To date, however, the functionality of the VDREs has not been verified in the context of the native CYP24 promoter, and there is no direct information available regarding the contribution of each VDRE to vitamin D induction or whether there is a cooperative interaction between the response elements. These issues are addressed in the current investigation, in which mutagenic constructs of the rat CYP24 promoter have been used in transient gene expression and gel mobility shift analysis. Construction of Mutant Clones-A 365-bp PvuII/StuI fragment containing 298 bp of 5Ј-flanking se...

Synergistic growth inhibition of prostate cancer cells by 1α,25 Dihydroxyvitamin D3 and its 19-nor-hexafluoride analogs in combination with either sodium butyrate or trichostatin A

“…A luciferase reporter construct containing the 5 half-site VDRE from the promoter/enhancer region of rat CYP24 (Kerry et al, 1996), (CYP24-Luc) was generated by cloning the sequence 5'-GATCAGAGCGCA CCCGCTGAACCC-TGCTGCCGGCGCCCTCACTCACCTCGCTGACTCCAT-G-3' (VDRE half sites in bold), synthesized as complementary oligonucleotides (Alta bioscience, Birmingham University, Birmingham, UK with BamHI compatible ends). Oligonucleotides were phosphorylated with ATP and T4 polynucleotide kinase (Pharmacia Biotech, St Albans, UK), complementary strands were annealed and cloned into a luciferase reporter vector containing a minimal TK promoter to give the ®nal pBLTKpA3LUC (CYP24-Luc).…”

Repression of basal transcription by vitamin D receptor: evidence for interaction of unliganded vitamin D receptor with two receptor interaction domains in RIP13delta1

“…Two oligonucleotide PCR primers, with KpnI sites, were employed to amplify 186 bp of CYP24 promoter sequence (encompassing a VDRE at 150/ 136 together with 74 bp of 5 -untranslated region) using as the template the plasmids pGL3WT ( 298) and pGL3M1 ( 298), the latter with a mutation in the proximal hexameric half site of the VDRE (5 -AGGTGA-3 to 5 -GCTTGA-3 ) as described by Kerry et al (1996). The PCR products for the wild type and mutant VDRE promoters were purified, digested with KpnI and ligated into the KpnI site of pGL3-Basic containing the firefly luciferase reporter gene (Promega, Madison, WI, USA), to generate pCYP24 WT-LUC and pCYP24M1-LUC.…”

“…In this study, we examine the regulation by 1,25-(OH) 2 D 3 of the gene for 25-hydroxyvitamin D 3 24-hydroxylase (CYP24) (Hahn et al 1994, Kerry et al 1996, the key enzyme involved with 1,25-(OH) 2 D 3 metabolism (Darwish & DeLuca 1996). In this communication, we have shown that VDR is involved in the repression of basal expression of a CYP24 promoter construct and also an unrelated promoter.…”

“…A double-stranded oligonucleotide (CYP24-VDRE) was synthesized that encompassed the VDRE at 150/ 136 in the rat CYP24 promoter (Kerry et al 1996) and contained SalI and XhoI overhangs at the 5 and 3 ends respectively as shown.…”

“…Cells were allowed to recover for 24 h and media replaced with RPMI medium containing 10% charcoal-stripped FCS. Cells were then further incubated or treated for 24 h with 10 7 M 1,25-(OH) 2 D 3 to activate CYP24 promoter expression, harvested in cell culture and lysis reagent (Promega) and luciferase activity determined as described previously (Kerry et al 1996).…”