The 3-epi- and 24-oxo-derivatives of 1alpha,25 dihydroxyvitamin D(3) stimulate transcription through the vitamin D receptor.

Vitamin D is enzymatically modified to more than 35 metabolites. While many of these are thought to represent degradation products, some have been shown to exhibit biological activity. We tested whether 3-epi-1alpha,25-dihydroxyvitamin D(3) (3-epi-1alpha, 25(OH)(2)D(3)), 1alpha,25-dihydroxy-24-oxo-vitamin D(3) (1alpha, 25(OH)(2)-24-oxo-D(3)), and 1alpha,25(OH)(2)D(3)-26,23-lactone can stimulate transcription of vitamin D responsive genes. MC3T3-E1 cells transfected with a 25-hydroxyvitamin D 24-hydroxylase (CYP24) promoter construct displayed a 6 fold response when treated with either 1alpha,25(OH)(2)D(3) or 3-epi-1alpha,25(OH)(2)D(3). Caco-2 cells were transfected with the wild type CYP24 promoter construct, or a Vitamin D Response Element (VDRE)-mutated form. Cells acquiring the wild type reporter responded to 1alpha,25(OH)(2)D(3) and 3-epi-1alpha,25(OH)(2)D(3) but not cells which acquired the mutated reporter. Additionally, VDR-negative COS-7 cells transfected with the wild type promoter responded (approximately 13 fold) to 1alpha, 25(OH)(2)D(3) and 3-epi-1alpha,25(OH)(2)D(3), only when co-transfected with the VDR. These results were confirmed using shorter incubation times and serum-free conditions. This strongly suggested that 3-epi-1alpha,25(OH)(2)D(3) mediates its effects through the VDR and its cognate binding site. Similar results were obtained with 1alpha,25(OH)(2)-24-oxo-D(3) using VDR-negative P19 cells. We could never detect activity from 1alpha,25(OH)(2)D(3)-26, 23-lactone on vitamin D-responsive target promoters. Our results firmly conclude that both 3-epi-1alpha,25(OH)(2)D(3) and the 1alpha, 25(OH)(2)-24-oxo-D(3) elicit their biological effects by acting through the VDR/VDRE.

The 25-hydroxyvitamin D 1-alpha-hydroxylase gene maps to the pseudovitamin D-deficiency rickets (PDDR) disease locus.

Pseudovitamin D-deficiency rickets (PDDR) is an autosomal recessive disorder that may be due to impaired activity of 25-hydroxyvitamin D-1alpha-hydroxylase, a renal cytochrome P450 enzyme (P450[1alpha]) of the vitamin D pathway. The disease locus for PDDR has been mapped by linkage analysis to 12q13-q14, but the molecular defect underlying the enzyme dysfunction has remained elusive due to the lack of sequence information for the P450(1alpha) gene (hereafter referred to as 1alpha-OHase). We have used a probe derived from the rat 25-hydroxyvitamin D-24-hydroxylase (CYP24; 24-OHase) sequence to identify and clone the 1alpha-OHase cDNA. The full-length 1alpha-OHase clone of 2.4 kb codes for a protein of predicted Mr 55 kDa. Functional activity of the cloned sequence was assessed using transient transfection, and the production of authentic 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] was confirmed using high performance liquid chromatography fractionation and time-of-flight mass spectrometry. The expression of the gene was analyzed in vitamin D-replete animals; treatment with 1alpha,25(OH)2D3 reduced 1alpha-OHase transcript levels by 70%, while administration of parathyroid hormone led to a 2-fold increase in the expression of the gene, thus confirming the hormonal regulation previously described using biochemical methods. The rat cDNA was used to obtain a human genomic clone. Interestingly, the human 1alpha-OHase gene mapped to 12q13.1-q13.3, providing strong evidence that a mutation in the 1alpha-OHase gene is responsible for the PDDR phenotype. The availability of a cloned sequence for 1alpha-OHase generates novel tools for the study of the molecular etiology of PDDR, and will allow the investigation of other disturbances of vitamin D metabolism.