The DNA f lanking the 5 sequence of the mouse 1␣-hydroxylase gene has been cloned and sequenced. A TATA box has been located at ؊30 bp and aCCAAT box has been located at ؊79 bp. The gene's promoter activity has been demonstrated by using a luciferase reporter gene construct transfected into a modified pig kidney cell line, AOK-B50. Parathyroid hormone stimulates this promoter-directed synthesis of luciferase by 17-fold, whereas forskolin stimulates it by 3-fold. The action of parathyroid hormone is concentration-dependent. 1,25-Dihydroxyvitamin D 3 does not suppress basal promoter activity and marginally suppresses parathyroid hormone-driven luciferase reporter activity. The promoter has three potential cAMP-responsive element sites, and two perfect and one imperfect AP-1 sites, while no DR-3 was detected. These results indicate that parathyroid hormone stimulates 25-hydroxyvitamin D 3 -1␣-hydroxylase by acting on the promoter of the 1␣-hydroxylase gene.Vitamin D is a major actor in calcium homeostasis of higher animals (1). To carry out these functions, vitamin D must be metabolized to its biologically active hormonal form, 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ). This is a two-step process requiring 25-hydroxylation in the liver and 1␣-hydroxylation in the kidney. The resulting hormone, 1,25-(OH) 2 D 3 , then binds to a nuclear vitamin D receptor (VDR) in target tissues, and the liganded receptor acts as a transcription factor to modulate the expression of specific genes encoding proteins that bring about the actions of vitamin D (2).The importance of the 1␣-hydroxylase enzyme is emphasized by the occurrence of a genetic disorder of vitamin D metabolism, vitamin D dependency rickets type I (VDDRI) (3). This disorder is characterized by very low serum 1,25-(OH) 2 D 3 levels despite normal vitamin D intakes (4) and is thought to be the result of a defect in the 1␣-hydroxylase gene (5). This link has recently been reinforced with the identification of the gene for human 1␣-hydroxylase (6). The gene was mapped to chromosomal region 12q13.1-q13.3, which contains the VDDRI disease locus.The 1␣-hydroxylation step is the most tightly regulated step in vitamin D metabolism. Several physiological factors interact to regulate 1␣-hydroxylase activity that, in turn, determines serum and tissue levels of 1,25-(OH) 2 D 3 . These regulators include parathyroid hormone (PTH) and hypophosphatemia, which stimulate 1␣-hydroxylase activity, and 1,25-(OH) 2 D 3 , which suppresses it (7-9).

It is now well established that supraphysiological doses of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] stimulate bone resorption. Recent studies have established that osteoblasts/stromal cells express receptor activator of NF-kappaB ligand (RANKL) in response to several bone-resorbing factors including 1alpha,25(OH)(2)D(3) to support osteoclast differentiation from their precursors. Osteoclast precursors which express receptor activator of NF-kappaB (RANK) recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage-colony stimulating factor (M-CSF). Osteoprotegerin (OPG) acts as a decoy receptor for RANKL. We also found that daily oral administration of 1alpha,25(OH)(2)D(3) for 14 days to normocalcemic thyroparathyroidectomized (TPTX) rats constantly infused with parathyroid hormone (PTH) inhibited the PTH-induced expression of RANKL and cathepsin K mRNA in bone. The inhibitory effect of 1alpha,25(OH)(2)D(3) on the PTH-induced expression of RANKL mRNA occurred only with physiological doses of the vitamin. Supraphysiological doses of 1alpha,25(OH)(2)D(3) increased serum Ca and expression of RANKL in vivo in the presence of PTH. These results suggest that the bone-resorbing activity of vitamin D does not occur at physiological dose levels in vivo. A certain range of physiological doses of 1alpha,25(OH)(2)D(3) rather suppress the PTH-induced bone resorption in vivo, supporting the concept that 1alpha,25(OH)(2)D(3) or its derivatives are useful for the treatment of various metabolic bone diseases such as osteoporosis and secondary hyperparathyroidism.

Two vitamin D-responsive elements (VDRE-1 and VDRE-2) were recently identified in the 5-upstream region of the rat 25-hydroxyvitamin D 3 24-hydroxylase gene at ؊151/؊137 and ؊259/؊245, respectively. We studied the transcriptional regulation of this gene by vitamin D by means of mutational analysis. Introducing mutations into VDRE-1 and VDRE-2 in the native promoter ؊291/؉9 reduced vitamin D-dependent chloramphenicol acetyltransferase activity by 86 and 41%, respectively. Mutation of the direct repeat ؊169/؊155 located at 3 base pairs upstream of VDRE-1 also caused 50% decrease of chloramphenicol acetyltransferase activity. Connection of the element ؊169/؊155 to VDRE-1 enhanced the vitamin D responsiveness of VDRE-1 5-fold through the heterologous ␤-globin promoter. The fragment ؊291/؊102 containing the two VDREs showed two shifted bands in the presence of the vitamin D receptor and retinoid X receptor in gel retardation analysis, and the appearance of the slower migrating band indicates that two sets of receptor complexes bind to this fragment simultaneously. These results demonstrate that VDRE-1 is a stronger mediator of vitamin D function than VDRE-2 due to the presence of the accessory element ؊169/؊155 located adjacent to VDRE-1, although VDRE-2 exhibits a smaller dissociation constant for the vitamin D receptor-retinoid X receptor complex than VDRE-1.Vitamin D 3 exerts its biological activity after conversion into 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 )1 by successive hydroxylations at C-25 in the liver and C-1 in the kidney. 1,25-(OH) 2 D 3 mediates various biological activities, including calcium homeostasis, bone remodeling, cell growth and differentiation, and immune responsiveness (1-5).One of the targets of 1,25-(OH) 2 D 3 is the vitamin D 24-hydroxylase gene. The enzyme expressed by this gene catalyzes 24-hydroxylation of 25-hydroxyvitamin D 3 and 1,25-(OH) 2 D 3 into 24,25-dihydroxyvitamin D 3 and 1,24,25-trihydroxyvitamin D 3 , respectively (6). 24-Hydroxylation is considered an inactivation process of 1,25-(OH) 2 D 3 (3, 7-9). We have isolated rat 24-hydroxylase cDNA and determined the gene structure (10, 11). The expression of 24-hydroxylase mRNA was strikingly increased by vitamin D in vivo and in vitro (9, 12, 13). Reinhardt and Horst (14) Three groups have independently identified two functional VDREs in an antisense orientation in the rat 24-hydroxylase gene promoter; at Ϫ151/Ϫ137 (VDRE-1) (24, 25) and Ϫ259/ Ϫ245 (VDRE-2) (26). Zierold et al. (27) examined the interaction between the two VDREs by means of a reporter gene assay using heterologous promoters and by a gel mobility shift assay. They concluded that the two VDREs synergistically increased binding affinity for the receptor complex to elicit powerful promoter activity.In this study, we assessed the functions of the two VDREs in more detail using a native promoter containing site-specific mutations at the VDREs and determined their affinity for the receptor complex by gel retardation assays. We found that the two VDREs work addi...

Osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoclast differentiation factor, inhibits both differentiation and function of osteoclasts. We previously reported that OPG-deficient mice exhibited severe osteoporosis caused by enhanced osteoclastic bone resorption. In the present study, potential roles of OPG in osteoclast differentiation were examined using a mouse coculture system of calvarial osteoblasts and bone marrow cells prepared from OPG-deficient mice. In the absence of bone-resorbing factors, no osteoclasts were formed in cocultures of wild-type (+/+) or heterozygous (+/-) mouse-derived osteoblasts with bone marrow cells prepared from homozygous (-/-) mice. In contrast, homozygous (-/-) mouse-derived osteoblasts strongly supported osteoclast formation in the cocultures with homozygous (-/-) bone marrow cells, even in the absence of bone-resorbing factors. Addition of OPG to the cocultures with osteoblasts and bone marrow cells derived from homozygous (-/-) mice completely inhibited spontaneously occurring osteoclast formation. Adding 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] to these cocultures significantly enhanced osteoclast differentiation. In addition, bone-resorbing activity in organ cultures of fetal long bones derived from homozygous (-/-) mice was markedly increased, irrespective of the presence and absence of bone-resorbing factors, in comparison with that from wild-type (+/+) mice. Osteoblasts prepared from homozygous (-/-), heterozygous (+/-), and wild-type (+/+) mice constitutively expressed similar levels of RANKL messenger RNA, which were equally increased by the treatment with 1alpha,25(OH)2D3. When homozygous (-/-) mouse-derived osteoblasts and hemopoietic cells were cocultured, but direct contact between them was prevented, no osteoclasts were formed, even in the presence of 1alpha,25(OH)2D3 and macrophage colony-stimulating factor. These findings suggest that OPG produced by osteoblasts/stromal cells is a physiologically important regulator in osteoclast differentiation and function and that RANKL expressed by osteoblasts functions as a membrane-associated form.